Three-dimensional (3D) microscopy has a broad range of applications from biomedical imaging to high-precision metrology. A wide range of optical methods offer the capability of imaging the 3D shape of a testing sample. For instance, microdeflectometry is a powerful noncontact tool modified from phase measuring deflectometry (PMD) for measuring nanometer defects on a freeform surface with large shape variation. A typical PMD system projects a fringe pattern in front of the surface under test and uses a camera to observe the fringe displacement reflected from the local surface. With the knowledge of the system geometry obtained from a rigorous process of calibration, surface gradient data are quantitatively measured and can be converted into surface shape via numerical integration. Microdeflectometry modifies the typical PMD system and uses a setup similar to the reflective light microscope, where the illumination module shares the optical path with the camera detection through the microscope objective and projects an aerial fringe image in front of the surface. By observing the fringe displacement, microdeflectometry measures small surface slope variation within a large angular range equivalent to the system numerical aperture with micron-level lateral resolution, which enables inspection of nanometer-sized defects within hundreds of microns of freeform surface height variation.
Structured illumination microscopy (SIM) is a widefield imaging technique in which structured illumination, e.g., a fringe pattern, is superimposed on the sample while capturing images. The fringe pattern is often shifted or rotated in steps between the capture of each image set. Though SIM has been used as an independent metrology tool, SIM has lower measuring stability for local variation than microdeflectometry.
Conventional 3D imaging techniques, such as microdeflectometry and SIM, are subject to the limit of a generally small depth of field (DOF) of standard microscopy systems. For instance, a microscope objective with a numerical aperture (NA) of 0.25 and a working wavelength of 0.5 μm has about ±8 μm DOF given by nλ/NA2, which theoretically is the half-width of the full DOF. (Here, λ is the wavelength of light imaged by the microscope objective and n is the index of refraction of the medium between the microscope objective and the sample.) Such a shallow DOF limits the capability of imaging a thick target or a surface with substantial height variation. Conventional extended depth-of-field (EDOF) microscopy extends the DOF by mounting the microscope objective or sample on a z-axis translation stage and taking measurements at different depths, with subsequent combination of focused data from the stack of images captured along the z-axis. However, the speed of z-axis translation must be kept low to prevent inertial vibration, which makes image capture a time-consuming process.
Researchers have developed a wide variety of techniques to accelerate the process of obtaining EDOF measurements. For instance, several non-scanning methods, including wavefront coding, chromatic methods, and volume holographic microscopy, have been demonstrated to obtain EDOF images in one single shot. These methods, however, either diminish depth information or provide very limited axial sampling, which is not applicable to high resolution three-dimensional measurements. Alternatively, another group of techniques improves scanning speed by integrating additional scanning devices that do not require mechanically moving the microscope objective or sample. One such example is a relay system with a scanning mirror for achieving remote scanning. Another example of non-mechanical scanning utilizes a commercially available focus-tunable lens for performing optical depth scanning. Among the commercial choices of focus-tunable lenses, the acoustic tunable lens (from, e.g., TAG Optics) provides a highest speed at 140 kHz. The electrically tunable lens (from, e.g., Optotune) provides only up to 400 Hz scanning rate but, on the other hand, supports stepwise scanning, retains system flexibility, and has the largest aperture for high power microscopic application. Successful implementations of an electrically tunable lens include multi-photon excitation microscopy, confocal microscopy, optical coherence tomography, photoacoustic microscopy, selective plane illumination microscopy, oblique back-illumination microscopy, and optical projection tomography.